Methods and compositions for stimulating neurogenesis and inhibiting neuronal degeneration using isothiazolopyrimidinones

ABSTRACT

The present invention provides methods and compositions comprising compounds useful for stimulating neurogenesis. The methods and compositions comprising compounds are also useful for inhibiting neuronal degeneration. Thus, the present invention can be used in the treatment of diseases and conditions characterized by neuronal loss and reduced neurogenesis including Alzheimer&#39;s disease, stroke, traumatic brain injury, traumatic nerve injury, and depression. This invention is useful for research products including single agents or mixtures of agents to promote, proliferate, differentiate, or maintain neurons from stem or progenitor cells.

PRIORITY

This application claims priority to U.S. Provisional Application No.60/945,524, filed Jun. 21, 2007.

FIELD OF INVENTION

The present invention generally relates to the field of neurology. Morespecifically, the present invention provides methods and compositionsfor stimulating neurogenesis and inhibiting neuronal degeneration.

BACKGROUND OF THE INVENTION

Alzheimer's disease is a brain disorder that gradually destroys neurons.Over 4.5 million people in America suffer from Alzheimer's, which mostlyoccurs in older adults. The risk of developing Alzheimer's diseaseapproximately doubles every five years after age 65 and reaches to 50percent by age 85. Patients afflicted with Alzheimer's disease losetheir ability to learn, remember, reason, make decisions, communicateand carry out daily activities. The direct and indirect cost of caringfor Alzheimer's disease patients has increased to at least $100 billionannually.

Stroke and traumatic brain injury can also cause neuronal loss and leadto cognitive decline.

The stimulation of neurogenesis may also be useful in treatingdepression. Depression is categorized by extreme changes in mood whichmay also be associated with psychoses. The association betweendepression, stress, and neurogenesis arose first from MRI imagingstudies suggesting a reduction in right and left hippocampal volumes inmajor depression (Sheline et al., 1996; Bremner et al., 2000; Mervaalaet al., 2000). Further research indicated that the volume loss in thebrain seen in patients with depression was due to glucocorticoid-inducedneuron loss specific to hippocampus (Lee et al., 2002 review; Lucassenet al., 2001; Sapolsky 2000).

Other studies further confirmed the close correlation betweenneurogenesis and depression. Data showed that chronic stress could causeboth volume changes and reduction in neurogenesis (Czeh et al., 2001;Pham et al., 2003). On the other hand, agents that cause a reduction inneurogenesis also appear as causative agents in depression specificallyglucocorticoids and depletion of serotonin (Brezun and Daszuta, 1999).Finally, research using X-rays to ablate new cells caused byfluoxetine-induced neurogenesis in mice could reverse the antidepressantbehavioral activity in the novelty suppressed feeding paradigm(Santerelli et al., 2003).

One challenge for using neurogenesis to treat Alzheimer's disease ordepression is that nascent neurons must still survive long enough toproduce functional neurons. There exists a need for a neurogenic agentthat promotes the proliferation of a neuronal precursor and that causesthe differentiation and survival of the neurons.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions comprisingcompounds useful for stimulating neurogenesis.

The methods and compositions of the present invention are particularlyuseful in the treatment of neurodegenerative disease like Alzheimer'sdisease and neuropsychiatric conditions such as depression. The methodsand compositions are useful for the manufacture of research productseither as one composition or as a mixture of compositions. The methodsand compositions comprising compounds are also useful for inhibitingneuronal degeneration. Thus, the present invention finds particularutility in the treatment of diseases and conditions characterized byneuronal loss including, but not limited to, Alzheimer's disease,stroke, traumatic brain injury, traumatic nerve injury and depression.Disclosed herein are the compounds, methods for making the compounds,compositions comprising the compounds, and methods for using thecompounds.

In one aspect, the present invention provides compositions comprisingcompounds useful for stimulating neurogenesis and/or inhibiting neuronaldegeneration. In a one aspect, a composition may comprise a compoundhaving the structure:

wherein R₁ is hydrogen, alkyl, branched alkyl, aryl, aralalkyl, benzyl,napthyl, cycloalkyl of 1 to 12 carbon atoms, or a heterocycle, when R₁is not hydrogen it can be optionally substituted with H, OH, alkyl,alkoxy, halogen, —CF₃, —R₂, —OR₂, —SR₂, —N(R₂)₂, —CN, —NO₂, —NC(O) R₂,—C(O) R₂, —C(O)N(R₂)₂, —S(O)₂R₂, —S(O)₂NR₂, —S(O)R₂, —C(O)R₂, —C(O)OR₂,or —C(O)N(R₂)₂ wherein each R₂ is independently H, alkyl, alkenyl,alkynyl, aryl, heterocycle, protecting group or prodrug moiety;

-   L is [CH₂]₂₋₆, —CR₂—(C═O)—CH₂—,

-   X is S, SO₂, O, or NH;-   Y is

-   Z is

each optionally substituted with H, OH, alkyl, alkoxy, halogen, or —CF₃;

-   T is O, S, or NR₁;-   n is 2 or 3;-   wherein each R₁ is independent and may be the same as each other or    preferably different.

In another aspect, the present invention is further directed to methodsand compositions comprising compounds that have utility in the treatmentof any diseases associated with neuron loss. More specifically, thepresent invention further provides methods for stimulating neurogenesisand/or inhibiting neuronal degeneration in a mammal. In another aspect,the method may comprise administering to a mammal a compositioncomprising a compound described herein. The composition comprising acompound described herein may be administered in an amount effective tostimulate neurogenesis and/or inhibit neuronal degeneration in themammal.

In a further aspect, a method for treating a mammal afflicted with aneurodegenerative disease or condition may comprise administering aneffective amount of a composition comprising a compound described hereinto the mammal.

In a further aspect, a method for treating a mammal afflicted with aneuropsychiatric disease or condition may comprise administering aneffective amount of a composition comprising a compound described hereinto the mammal. In another aspect, the mammal is a human.

In another aspect, the human is a patient suffering from a conditionselected from the neurodegenerative disease, brain injury, nerve injury,psychiatric disorders and aging.

In yet another aspect, the present invention also comprisespharmaceutical compositions comprising the compounds disclosed herein.Routes of administration and dosages of effective amounts of thepharmaceutical compositions comprising the compounds are also disclosed.The compounds of the present invention can be administered incombination with other pharmaceutical agents in a variety of protocolsfor effective treatment of disease.

The invention includes methods for the administration of pharmaceuticalcompositions comprising the compounds disclosed herein or a number ofcompounds together, including the use of said compounds in conjunctionwith other drugs and/or cell therapies to promote neurogenesis and/orneuroprotection. The present invention includes methods ofadministration and compositions comprising prodrug forms of the activeingredients and their transition forms.

The invention additionally includes kits comprising the compounds andcompositions of the invention, as a means to provide standardizedreagents and medicaments, as required by current clinical practice, asknown in the art. The kits of the invention include testing andscreening kits and methods, to enable practitioners to measure levels ofthe active ingredients in bodily fluids. The kits of the invention alsoinclude research-grade reagents and kits available for use and purchaseby research entities.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methodologies, assays, etc. described herein, as these mayvary. It is also to be understood that the terminology used herein isused for the purpose of describing exemplary aspects of the invention,and is not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Preferred methods andcompositions are described, although any methods and compositionssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention.

I. DEFINITIONS

As used herein, the term “compound” refers to all of the iterations ofthe structure and formula disclosed herein and also includes a referenceto a physiologically acceptable salt thereof. Examples ofphysiologically acceptable salts of the compounds of the presentinvention include salts derived from an appropriate base, such as analkali metal, such as sodium, and alkaline earth, such as magnesium,ammonium and NX₄ ⁺ (wherein X is C₁-C₄ alkyl). Physiologicallyacceptable salts of a hydrogen atom or an amino group may include, butare not limited to, salts of organic carboxylic acids such as acetic,benzoic, lactic, fumaric, tartaric, maleic, malonic, malic, isethionic,lactobionic and succinic acids; organic sulfonic acids, such asmethanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonicacids; and inorganic acids, such as hydrochloric, sulfuric, phosphoricand sulfamic acids. Physiologically acceptable salts of a compound of ahydroxy group include, but are not limited to, the anion of the compoundin combination with a suitable cation such as Na⁺ and NX₄ ⁺ (wherein Xis independently selected from H or a C₁-C₄ alkyl group).

For therapeutic use, salts of the compounds of the present inventionwill be physiologically acceptable, i.e., the salts will be derived froma physiologically acceptable acid or base. Salts of acids or bases,however, which are not physiologically acceptable, may also find use inthe preparation or purification of a physiologically acceptablecompound. Thus, all salts, whether or not derived from a physiologicallyacceptable acid or base, are within the scope of the present invention.Also included within this invention are pharmaceutically acceptablesolvates and hydrates of the compounds.

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto, ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH══CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH══CH₂).

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e., acarbon-carbon, sp triple bond. Examples include, but are not limited to,acetylenic (—C≡CH) and propargyl (—CH₂C≡CH).

The terms “alkylene” and “alkyldiyl” each refer to a saturated, branchedor straight chain or cyclic hydrocarbon radical of 1-18 carbon atoms,and having two monovalent radical centers derived by the removal of twohydrogen atoms from the same or two different carbon atoms of a parentalkane. Typical alkylene radicals include, but are not limited to,methylene (—CH₂—) 1,2-ethyl (—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—),1,4-butyl (—CH₂CH₂CH₂CH₂—), and the like. “Alkenylene” refers to anunsaturated, branched or straight chain or cyclic hydrocarbon radical of2-18 carbon atoms, and having two monovalent radical centers derived bythe removal of two hydrogen atoms from the same or two different carbonatoms of a parent alkene, i.e., double carbon-carbon bond moiety.Typical alkenylene radicals include, but are not limited to,1,2-ethylene (—CH══CH—).

“Alkynylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne, i.e.,triple carbon-carbon bond moiety. Typical alkynylene radicals include,but are not limited to, acetylene (—C≡C—), propargyl (—CH₂C≡C—), and4-pentynyl (—CH₂CH₂CH₂C≡CH—).

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like.

“Heteroaryl” means a monovalent aromatic radical of one or more carbonatoms and one or more atoms selected from N, O, S, or P, derived by theremoval of one hydrogen atom from a single atom of a parent aromaticring system. Heteroaryl groups may be a monocycle having 3 to 7 ringmembers (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O,P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atomsand 1 to 3 heteroatoms selected from N, O, P, and S). Heteroarylbicycles have 7 to 10 ring atoms (6 to 9 carbon atoms and 1 to 2heteroatoms selected from N, O, and S) arranged as a bicyclo [4,5],[5,5], [5,6], or [6,6] system; or 9 to 10 ring atoms (8 to 9 carbonatoms and 1 to 2 hetero atoms selected from N and S) arranged as abicyclo [5,6] or [6,6] system. The heteroaryl group may be bonded to thedrug scaffold through a carbon, nitrogen, sulfur, phosphorus or otheratom by a stable covalent bond. Heteroaryl groups include pyridyl,dihydropyridyl isomers, pyridazinyl, pyrimidinyl, pyrazinyl,s-triazinyl, oxazolyl, imidazolyl, thiazolyl, isoxazolyl, pyrazolyl,isothiazolyl, furanyl, thiofuranyl, thienyl, and pyrrolyl.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., thealkyl moiety, including alkanyl, alkenyl or alkynyl groups, of thearylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14carbon atoms.

Substituted substituents such as “substituted alkyl,” “substitutedaryl,” “substituted heteroaryl,” and “substituted arylalkyl” mean alkyl,aryl, and arylalkyl respectively, in which one or more hydrogen atomsare each independently replaced with a substituent. Typical substituentsinclude, but are not limited to, —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃,══NR, —CX3, —CN, —OCN, —SCN, —N══C══O, —NCS, —NO, —NO₂, ══N₂, —N₃,NC(══O)R, —C(══O)R, —C(══O)NRR, —S(══O)₂O⁻, —S(══O)₂OH, —S(══O)₂R,—OS(══O)₂OR, —S(═O₂NR, —S(══O)R, —OP(══O)O₂RR, —P(══O)O₂RR,—P(══O)(O⁻)₂, —P(══O)(OH)₂, —C(══O)R, —C(══O)X, —C(S)R, —C(O)OR,—C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR, —C(NR)NRR, whereeach X is independently a halogen: F, Cl, Br, or I; and each R isindependently —H, alkyl, aryl, heterocycle, protecting group or prodrugmoiety. Alkylene, alkenylene, and alkynylene groups may also besimilarly substituted.

“Halogens” includes F, Cl, Br or I and is used interchangeably with theword “halo.”

“Heterocycle” means a saturated, unsaturated or aromatic ring systemincluding at least one N, O, S, or P. Heterocycle thus includesheteroaryl groups. Heterocycle as used herein includes, but is notlimited to heterocycles described in PAQUETTE, PRINCIPLES OF MODERNHETEROCYCLIC CHEMISTRY (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; THE CHEMISTRY OF HETEROCYCLIC COMPOUNDS,A SERIES OF MONOGRAPHS (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; KATRITZKY ET AL.,COMPREHENSIVE HETEROCYCLIC CHEMISTRY (Pergamon Press, 1996); and 82 J.AM. CHEM. SOC. 5566 (1960).

Heterocycles include, but are not limited to pyridyl, dihydroypyridyl,tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfuroxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

Carbon bonded heterocycles include but are not limited to those that arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

Nitrogen bonded heterocycles include but are not limited to those thatare bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole,position 2 of a isoindole, or isoindoline, position 4 of a morpholine,and position 9 of a carbazole, or β-carboline. Still more typically,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

“Carbocycle” means a saturated, unsaturated or aromatic ring systemhaving 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as abicycle. Monocyclic carbocycles have 3 to 6 ring atoms, still moretypically 5 or 6 ring atoms. Bicyclic carbocycles have 7 to 12 ringatoms, e.g., arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system,or 9 or 10 ring atoms arranged as a bicyclo [5,6] or [6,6] system.Monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl, spiryland naphthyl. Carbocycle thus includes aryl groups.

As used herein, the term “chiral” refers to molecules which have theproperty of non-superimposability of the mirror image partner, while theterm “achiral” refers to molecules which are superimposable on theirmirror image partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followMCGRAW-HILL DICTIONARY OF CHEMICAL TERMS (S. P. Parker, Ed., McGraw-HillBook Company, New York, 1984); and ELIEL, E. AND WILEN, S.,STEREOCHEMISTRY OF ORGANIC COMPOUNDS (John Wiley & Sons, Inc., New York,1994). Many organic compounds exist in optically active forms, i.e.,they have the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and 1 or (+) and (−) are employed todesignate the sign of rotation of plane-polarized light by the compound,with (−) or 1 meaning that the compound is levorotatory. A compoundprefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The terms “treatment,” “treating,” “treat,” “therapy,” “therapeutic,”and the like are used herein to refer generally to obtaining a desiredpharmacological and/or physiological effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete stabilization or cure for a disease and/or adverse effectattributable to the disease. “Treatment” as used herein covers anytreatment of a disease in a subject, and includes: (a) preventing thedisease or symptom from occurring in a subject which may be predisposedto the disease or symptom, but has not yet been diagnosed as having it;(b) inhibiting the disease symptom, i.e., arresting its development; or(c) relieving the disease symptom, i.e., causing regression of thedisease or symptom.

The term “pharmaceutically acceptable carrier,” as used herein, refersto any and all solvents, dispersion media, coatings, antibacterial andantifungal agent, isotonic and absorption delaying agents forpharmaceutical active substances as are well known in the art. Exceptinsofar as any conventional media or agent is incompatible with thecompound, its use in the therapeutic compositions is contemplated.Supplementary compounds can also be incorporated into the compositions.

As used herein, the term “excipient” refers to the additives used toconvert an active compound into a form suitable for its intendedpurpose. For compositions of the present invention suitable foradministration to a human, the term “excipient” includes thoseexcipients described in the HANDBOOK OF PHARMACEUTICAL EXCIPIENTS,American Pharmaceutical Association, 2nd Ed. (1994), which is hereinincorporated in its entirety. The term “excipients” is meant to includefillers, binders, disintegrating agents, lubricants, solvents,suspending agents, dyes, extenders, surfactants, auxiliaries and thelike. Liquid excipients can be selected from various oils, includingthose of petroleum, animal, vegetable or synthetic origin, such as,peanut oil, soybean oil, mineral oil, sesame oil, hydrogenated vegetableoil, cottonseed oil, groundnut oils, corn oil, germ oil, olive oil, orcastor oil, and so forth.

Suitable excipients also include, but are not limited to, fillers suchas saccharides, lactose, fructose, sucrose, inositol, mannitol orsorbitol, xylitol, trehalose, cellulose preparations and/or calciumphosphates, tricalcium phosphate or calcium hydrogen phosphate, as wellas starch paste, using modified starch, maize starch, wheat starch, ricestarch, potato starch, gelatin, tragacanth, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, hydroxypropyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, aluminum metahydroxide, bentonite, sodiumcarboxymethylcellulose, croscarmellose sodium, crospovidone and sodiumstarch glycolate, and/or polyvinyl pyrrolidine and mixtures thereof. Ifdesired, disintegrating agents can be added, such as, theabove-mentioned starches and also carboxymethyl-starch, cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as,sodium alginate. Auxiliaries include, silica, stearic acid or saltsthereof, such as, magnesium stearate, sodium stearyl fumarate, orcalcium stearate.

The expression “therapeutically effective amount” refers to an amount ofa compound disclosed herein, that is effective for preventing,ameliorating, treating or delaying the onset of a disease or condition.

The pharmaceutical compositions of the inventions can be administered toany animal that can experience the beneficial effects of the compoundsof the invention. Such animals include humans and non-humans such asprimates, pets and farm animals.

II. DESCRIPTION OF THE COMPOUNDS

In one aspect, the present invention includes compositions comprising acompound having the structure:

wherein R₁ is hydrogen, alkyl, branched alkyl, aryl, aralalkyl, benzyl,napthyl, cycloalkyl of 1 to 12 carbon atoms, or a heterocycle, when R₁is not hydrogen it can be optionally substituted with H, OH, alkyl,alkoxy, halogen, —CF₃, —R₂, —OR₂, —SR₂, —N(R₂)₂, —CN, —NO₂, —NC(O)R₂,—C(O)R₂, —C(O)N(R₂)₂, —S(O)₂R₂, —S(O)₂NR₂, —S(O)R₂, —C(O)R₂, —C(O)OR₂,or —C(O)N(R₂)₂ wherein each R₂ is independently H, alkyl, alkenyl,alkynyl, aryl, heterocycle, protecting group or prodrug moiety;

-   L is [CH₂]₂₋₆, —CH₂—(C═O)—CH₂—,

-   X is S, SO₂, O, or NH;-   Y is

-   Z is

each optionally substituted with H, OH, alkyl, alkoxy, halogen, or —CF₃;

-   T is O, S, or NR₁;-   n is 2 or 3;-   wherein each R₁ is independent and may be the same as each other or    preferably different.

Additional aspects of the invention include compounds of the followingformulas and compositions comprising said compounds:

TABLE 1 STRUCTURE NAME

3-(4-Methoxyphenyl)-6-[(1-ethylpyrrolidin-2-yl)methyl]-isothiazolo[4,5-d]pyrimidin- 7(6H)-one

3-(4-Methoxyphenyl)-6-[3-(4- morpholinyl)propyl]-isothiazolo[4,5-d]pyrimidin-7(6H)-one

3-(4-chlorophenyl)-6-(3-phenylpropyl)-isothiazolo[4,5-d]pyrimidin-7(6H)-one

3-(4-Methoxyphenyl)-6-[3-(2-oxopyrrolidin-1-yl)propyl]-isothiazolo[4,5-d]pyrimidin- 7(6H)-one

3-(4-Chlorophenyl)-6-[3-(4- morpholinyl)propyl]isothiazolo[4,5-d]pyrimidin-7(6H)-one

3-(2-Fluoro-4-methoxyphenyl)-6-[3-(4-morpholinyl)propyl[-isothiazolo[4,5- d]pyrimidin-7(6H)-one

3-(4-methoxyphenyl)-isothiazolo[4,5-d]pyrimidin-7(6H)-on-6-yl-methylamino, morpholinyl urea

3-(4-methoxyphenyl)-isothiazolo[4,5- d]pyrimidin-7(6H)-on-6-yl, 4-[(4-fluorophenyl)piperidinyl]urea

6-((1-ethylpyrrolidin-2-yl)methyl)-3-(4-methoxyphenyl)isothiazolo[4,5-d]pyrimidin- 7(6H)-one

6-((1-ethylpyrrolidin-2-yl)methyl)-3-(4- fluorophenyl)isothiazolo[4,5-d]pyrimidin- 7(6H)-one

3-(4-methoxyphenyl)-6-((1-methylpiperidin-2-yl)methyl)isothiazolo[4,5-d]pyrimidin- 7(6H)-one

Finally, the general structure of the compounds of the present inventionmay encompass all states of saturation of the substituents shown, suchas all ene, diene, triene, and yne derivatives of any substituent. Thegeneral structures also encompass all conformational isomers,regioisomers, and stereoisomers that may arise from a particular set ofsubstituents. The general structures also encompass all enantiomers,diastereomers, and other optical isomers whether in enantiomeric orracemic forms, or mixtures of stereoisomers.

III. COMPOSITIONS COMPRISING COMPOUNDS OF THE INVENTION

The present invention also comprises pharmaceutical compositionscomprising the compounds disclosed herein. Routes of administration anddosages of effective amounts of the pharmaceutical compositionscomprising the compounds are also disclosed. The compounds of thepresent invention can be administered in combination with otherpharmaceutical agents in a variety of protocols for effective treatmentof disease.

The pharmaceutical compositions of the inventions can be administered toany animal that can experience the beneficial effects of the compoundsof the invention. Such animals include humans and non-humans such aspets and farm animals.

The pharmaceutical compositions of the present invention areadministered to a subject in a manner known in the art. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

In addition to the compounds disclosed herein, the pharmaceuticalcompositions of the present invention may further comprise at least oneof any suitable auxiliaries including, but not limited to, diluents,binders, stabilizers, buffers, salts, lipophilic solvents,preservatives, adjuvants or the like. Pharmaceutically acceptableauxiliaries are preferred. Examples and methods of preparing suchsterile solutions are well known in the art and can be found in wellknown texts such as, but not limited to, REMINGTON'S PHARMACEUTICALSCIENCES (Gennaro, Ed., 18th Edition, Mack Publishing Co. (1990)).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofthe compound.

Pharmaceutical excipients and additives useful in the present inventioncan also include, but are not limited to, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination in ranges of 1-99.99%by weight or volume. Exemplary protein excipients include serum albuminsuch as human serum albumin (HSA), recombinant human albumin (rHA),gelatin, casein, and the like. Representative amino acid components,which can also function in a buffering capacity, include alanine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like.

Carbohydrate excipients suitable for use in the present inventioninclude monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), myoinositol and the like.

The composition further can contain, but is not limited topharmaceutically acceptable carriers such as coloring agents,emulsifying agents, suspending agents, ethanol, EDTA, citrate buffer,flavoring, and water.

The composition of the invention also can contain the preservativesmethylparaben (also known as 4-hydroxybenzoic acid methyl ester; methylp-hydroxybenzoate; or METHYL CHEMOSEPT), ethylparaben (also known as4-hydroxybenzoic acid ethyl ester; ethyl p-hydroxybenzoate; or ETHYLPARASEPT), propylparaben (also known as 4-hydroxybenzoic acid propylester; propyl p-hydroxybenzoate; NIPASOL; or PROPYL CHEMOSEPT) and/orbutylparaben (also known as 4-hydroxybenzoic acid propyl ester; propylp-hydroxybenzoate; or BUTYL CHEMOSEPT). In some aspects, the compositioncontains methylparaben and/or propylparaben.

Emulsifiers of the invention include, but are not limited to ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

The pharmaceutical compositions comprising the compounds of the presentinvention can also include a buffer or a pH adjusting agent. Typically,the buffer is a salt prepared from an organic acid or base.Representative buffers include organic acid salts such as salts ofcitric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid,succinic acid, acetic acid, or phthalic acid; Tris, tromethaminehydrochloride, or phosphate buffers.

Additionally, the pharmaceutical compositions of the invention caninclude polymeric excipients/additives such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin), polyethylene glycols, flavoringagents, anti-microbial agents, sweeteners, antioxidants, anti-staticagents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g.,cholesterol), and chelating agents (e.g., EDTA or EGTA). These andadditional known pharmaceutical excipients and/or additives suitable foruse in the present invention are known in the art, e.g., as listed inREMINGTON: THE SCIENCE & PRACTICE OF PHARMACY (19^(th) ed., Williams &Williams (1995)) and PHYSICIAN'S DESK REFERENCE (52^(nd) ed., MedicalEconomics (1998)), the disclosures of which are expressly entirelyincorporated herein by reference.

The present invention provides stable pharmaceutical compositions aswell as preserved solutions and compositions containing a preservative,as well as multi-use preserved compositions suitable for pharmaceuticalor veterinary use, comprising at least one compound disclosed herein ina pharmaceutically acceptable composition. Pharmaceutical compositionsin accordance with the present invention may optionally contain at leastone known preservative. Preservatives include, but are not limited to,phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol,magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl,propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof inan aqueous diluent. Any suitable concentration or mixture can be used asknown in the art, such as 0.001-5%, or any range or value therein.Non-limiting examples include, no preservative, 0.1-2% m-cresol, 0.1-3%benzyl alcohol, 0.001-0.5% thimerosal, 0.001-2.0% pheno, 0.0005-1.0%alkylparaben(s), and the like.

Other excipients, e.g., isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally added to the diluent. Anisotonicity agent such as glycerin, is commonly used at knownconcentrations. A physiologically tolerated buffer is preferably addedto provide improved pH control. The pharmaceutical compositions cancover a wide range of pHs, such as from about pH 4 to about pH 10,specifically, a range from about pH 5 to about pH 9, and morespecifically, a range of about 6.0 to about 8.0. In one aspect, theformulations of the present invention have pH between about 6.8 andabout 7.8. Suitable buffers include phosphate buffers, sodium phosphateand phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), TWEEN 40(polyoxyethylene (20) sorbitan monopalmitate), TWEEN 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, PLURONIC® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the pharmaceutical compositionsto reduce aggregation. These additives are particularly useful if a pumpor plastic container is used to administer the pharmaceuticalcomposition. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the composition to aggregate.

The composition of the invention also can contain the preservativesmethylparaben (also known as 4-hydroxybenzoic acid methyl ester; methylp-hydroxybenzoate; or METHYL CHEMOSEPT), ethylparaben (also known as4-hydroxybenzoic acid ethyl ester; ethyl p-hydroxybenzoate; or ETHYLPARASEPT), propylparaben (also known as 4-hydroxybenzoic acid propylester; propyl p-hydroxybenzoate; NIPASOL; or PROPYL CHEMOSEPT) and/orbutylparaben (also known as 4-hydroxybenzoic acid propyl ester; propylp-hydroxybenzoate; or BUTYL CHEMOSEPT). In some aspects, the compositioncontains methylparaben and/or propylparaben.

The compositions of the present invention can also be administered inthe form of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto the compounds of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art (see Prescott, ed., METH.CELL BIOL. 14:33 (1976)). Liposomes, methods of making and methods ofuse are described in U.S. Pat. No. 4,089,8091 (process for thepreparation of liposomes), U.S. Pat. No. 4,233,871 (methods regardingbiologically active materials in lipid vescisles), U.S. Pat. No.4,438,052 (process for producing mixed miscelles), U.S. Pat. No.4,485,054 (large multilamellar vescisles), U.S. Pat. No. 4,532,089(giant-sized liposomes and methods thereof), U.S. Pat. No. 4,897,269(liposomal drug delivery system), U.S. Pat. No. 5,820,880 (liposomalformulations), and so forth.

During any of the processes for preparation of the compounds of thepresent invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in PROTECTIVE GROUPS IN ORGANIC CHEMISTRY (1973); and GREENEAND WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (1991). The protectinggroups may be removed at a convenient subsequent stage using methodsknown from the art.

The compound of the invention can be solubilized or suspended in apreconcentrate (before dilutions with a diluent), added to thepreconcentrate prior to dilution, added to the diluted preconcentrate,or added to a diluent prior to mixing with the preconcentrate. Thecompound of the invention can also be co-administered as part of anindependent dosage form, for therapeutic effect. Optionally, thecompound of the invention can be present in a first, solubilized amount,and a second, non-solubilized (suspended) amount.

The pharmaceutical formulation can also contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations that can be administered to animals, as described herein.

For oral administration in the form of a tablet or capsule, a compoundmay be combined with an oral, non-toxic pharmaceutically acceptableinert carrier such as ethanol, glycerol, water and the like. Moreover,when desired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents may also be incorporated into the mixture.Suitable binders include, without limitation, starch; gelatin; naturalsugars such as glucose or beta-lactose; corn sweeteners; natural andsynthetic gums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose; polyethylene glycol; waxes and the like.Lubricants used in these dosage forms include, without limitation,sodium oleate, sodium stearate, magnesium stearate, sodium benzoate,sodium acetate, sodium chloride and the like. Disintegrators include,without limitation, starch, methyl cellulose, agar, bentonite, xanthangum and the like.

For oral administration, the composition also optionally contains asweetener. Sweeteners include but are not limited to sucrose, fructose,sodium saccharin, sucralose (SPLENDA®), sorbitol, mannitol, aspartame,sodium cyclamate, and the like and combinations thereof.

The aqueous suspensions, emulsions and/or elixirs for oraladministration of this invention can be combined with various sweeteningagents, flavoring agents, such as, but not limited to orange or lemonflavors, coloring agents, such as dye stuffs, natural coloring agents orpigments, in addition to the diluents such as water, glycerin andvarious combinations, as described herein.

The pharmaceutical compositions of the present invention suitable fororal administration may be presented as discrete units such as capsules,dragees, cachets or tablets each containing a predetermined amount ofthe compound; as a powder or granules; as a solution or a suspension inan aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil emulsion, and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the compound in a free-flowing formsuch as a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding, in a suitable machine, a mixture of thepowdered compound moistened with an inert liquid diluent. The tabletsmay be optionally coated or scored and may be formulated so as toprovide a slow or controlled release of the compound therein.

In addition, the compositions comprising compounds may be incorporatedinto biodegradable polymers allowing for sustained release of thecompound. The biodegradable polymers and their uses are described indetail in Brem et al., 74 J. NEUROSURG. 441-46 (1991). Suitable examplesof sustained-release compositions include semipermeable matrices ofsolid hydrophobic polymers containing a compound of the presentinvention, which matrices are in the form of shaped articles, e.g.,films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (including poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT® (Tap Pharmaceuticals, Inc., Chicago, Ill.)(injectable microspheres composed of lactic acid glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

Pharmaceutical compositions suitable for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes that renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

For parenteral administration, sterile suspensions and solutions aredesired. Isotonic preparations which generally contain suitablepreservatives are employed when intravenous administration is desired.The pharmaceutical compositions may be administered parenterally viainjection of a pharmaceutical composition comprising a compounddissolved in an inert liquid carrier. The term “parenteral,” as usedherein, includes, but is not limited to, subcutaneous injections,intravenous, intramuscular, intraperitoneal injections, or infusiontechniques. Acceptable liquid carriers include, vegetable oils such aspeanut oil, cotton seed oil, sesame oil and the like, as well as organicsolvents such as solketal, glycerol formal and the like. Thepharmaceutical compositions may be prepared by dissolving or suspendingthe compound in the liquid carrier such that the final formulationcontains from about 0.005% to 30% by weight of a compound.

The composition of the invention can also include additional therapeuticagents such as, but not limited to hydrophilic drugs, hydrophobic drugs,hydrophilic macromolecules, cytokines, peptidomimetics, peptides,proteins, toxoids, sera, antibodies, vaccines, nucleosides, nucleotides,nucleoside analogs, genetic materials and/or combinations thereof.

Examples of therapeutic agents that can be used in the pharmaceuticalcompositions of the present invention include, but are not limited to,other antineoplastic agents, analgesics and anti-inflammatory agents,anti-anginal agents, antihelmintics, anti arrythmic agents,anti-arthritic agents, anti-asthma agents, anti-bacterial agents,anti-viral agents, antibiotics, anti-coagulants, anti-depressants,anti-diabetic agents, anti-epileptic agents, anti-emetics, anti-fungalagents, anti-gout agents, anti-hypertensive agents, anti-malarialagents, antimigraine agents, anti-muscarinic agents, anti-parkinson'sagents, anti-protozoal agents, anti-thyroid agents, thyroid therapeuticagents, anti-tussives, anxiolytic agents, hypnotic agents, neurolepticagents, n-blockers, cardiac inotropic agents, corticosteroids,diuretics, gastrointestinal agents, histamine H-receptors antagonists,immunosuppressants, keratolytics, lipid regulating agents, musclerelaxants, nutritional agents, cytokines, peptidomimetics, peptides,proteins, toxoids, sera, sedatives, sex hormones, sex hormoneantagonists or agonists, stimulants antibodies, vaccines, nucleosides,nucleoside analogs and genetic materials. Amphiphilic therapeutic agentsand nutritional agents can also be included.

The additional therapeutic agent can be solubilized or suspended in apreconcentrate (before dilutions with a diluent), added to thepreconcentrate prior to dilution, added to the diluted preconcentrate,or added to a diluent prior to mixing with the preconcentrate. Theadditional therapeutic agent can also be co-administered as part of anindependent dosage form, for therapeutic effect. Optionally, theadditional therapeutic agent(s) can be present in a first, solubilizedamount, and a second, non-solubilized (suspended) amount. Suchadditional therapeutic agent(s) can be any agent(s) having therapeuticor other value when administered to an animal, particularly to a mammal,such as drugs, nutrients, and diagnostic agents.

In addition to the compound and compositions of the invention, andadditional pharmaceutically active agents, the pharmaceuticalformulation can also contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries that facilitateprocessing of the active compounds into preparations that can beadministered to animals, as described herein.

Pharmaceutical formulations useful in the present invention can containa quantity of a compound(s) according to this invention in an amounteffective to treat the condition, disorder or disease of the subjectbeing treated.

The invention is also directed to a kit form useful for administrationto patients in need thereof. The kit may have a carrier means beingcompartmentalized in close confinement to receive two or more containermeans therein, having a first container means containing atherapeutically effective amount of a pharmaceutical composition of theinvention and a carrier, excipient or diluent. Optionally, the kit canhave additional container mean(s) comprising a therapeutically effectiveamount of additional agents.

The kit comprises a container for the separate compositions such as adivided bottle or a divided foil packet, however, the separatecompositions can also be contained within a single, undivided container.Typically, the kit contains directions for administration of theseparate components. The kit form is particularly advantageous when theseparate components are preferably administered at different dosageintervals, or when titration of the individual components of thecombination is desired by the prescribing physician. The kits of theinvention include testing and screening kits and methods, to enablepractitioners to measure levels of the active ingredients in bodilyfluids. The kits of the invention also include research-grade reagentsand kits available for use and purchase by research entities.

IV. ROUTES OF ADMINISTRATION OF COMPOSITIONS COMPRISING THE COMPOUNDS OFTHE INVENTION

The invention further relates to the administration of at least onecompound disclosed herein by the following routes, including, but notlimited to oral, parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarenal, intraretinal, intraspinal, intrasynovial,intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,buccal, sublingual, intranasal, iontophoretic means, or transdermalmeans.

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for periods ofone week to one year from a single administration. Certain medicaldevices may be employed to provide a continuous intermittent or ondemand dosing of a patient. The devices may be a pump of diffusionapparatus, or other device containing a reservoir of drug and optionallydiagnostic or monitoring components to regulate the delivery of thedrug. Various slow-release, depot or implant dosage forms can beutilized. A dosage form can contain a pharmaceutically acceptablenon-toxic salt of a compound disclosed herein that has a low degree ofsolubility in body fluids, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g., a zinc tannate salt. Additionally, the compounds ofthe present invention or a relatively insoluble salt such as those justdescribed, can be formulated in a gel, an aluminum monostearate gelwith, e.g., sesame oil, suitable for injection. Salts include, but arenot limited to, zinc salts, zinc tannate salts, pamoate salts, and thelike. Another type of slow-release depot formulation for injection wouldcontain the compound or salt dispersed or encapsulated in a slowdegrading, non-toxic, non-antigenic polymer such as a polylacticacid/polyglycolic acid polymer, including the formulations as describedin U.S. Pat. No. 3,773,919. The compounds or relatively insoluble saltsthereof such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow-release, depot or implant formulations, e.g., gas orliquid liposomes are known in the literature. See, e.g., U.S. Pat. No.5,770,222; SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS(1978).

Other examples include provision of the compounds of the presentinvention to be administered by sustained release delivery systemcontaining a biodegradable composition. The biodegradable compositionmay be composed of a biodegradable, water-coagulable, non-polymericmaterial and a biocompatible, non-toxic organic solvent that is miscibleto dispersible in an aqueous medium. The delivery system may beimplanted at an implant site causing the solvent to dissipate, disperseor leach from the composition into surrounding tissue fluid through aresulting microporous matrix.

The term “implant site” is meant to include a site, in or on which thenon-polymeric composition is applied. Implantation or implant site canalso include the incorporation of the pharmaceutical compositioncomprising at least one compound of the present invention with a soliddevice. The pharmaceutical composition can be incorporated into acoating on a stent that is implanted into a subject. Additionally, othersolid or biodegradeable materials can be used as a substrate on whichthe pharmaceutical composition is applied. The coated material,comprising the pharmaceutical composition is then implanted, inserted oris adjacent to the subject or patient. The term “biodegradable” meansthat the non-polymeric material and/or matrix of the implant willdegrade over time by the action of enzymes, by simple or enzymaticallycatalyzed hydrolytic action and/or by other similar mechanisms in thehuman body. By “bioerodible,” it is meant that the implant matrix willerode or degrade over time due, at least in part, to contact withsubstances found in the surrounding tissue fluids, cellular action, andthe like. By “bioabsorbable,” it is meant that the non-polymeric matrixwill be broken down and absorbed within the human body, by a cell, atissue, and the like.

Non-polymeric materials that can be used in the composition generallyare those that are biocompatible, substantially insoluble in water andbody fluids, and biodegradable and/or bioerodible. The non-polymericmaterial is capable of being at least partially solubilized in awater-soluble organic solvent. The non-polymeric materials are alsocapable of coagulating or solidifying to form a solid implant matrix.The non-polymeric material is combined with a compatible and suitableorganic solvent to form a composition that has the desired consistencyranging from watery to viscous to a spreadable putty or paste.

Suitable organic solvents are those that are biocompatible,pharmaceutically-acceptable, and will at least partially dissolve thenon-polymeric material. The organic solvent has a solubility in waterranging from miscible to dispersible. Optionally, a pore-forming agentcan be included in the composition to generate additional pores in theimplant matrix. The pore-forming agent can be any organic or inorganic,pharmaceutically-acceptable substance that is substantially soluble inwater or body fluid, and will dissipate from the coagulatingnon-polymeric material and/or the solid matrix of the implant intosurrounding body fluid at the implant site.

The compounds of the present invention are capable of providing a localor systemic biological, physiological or therapeutic effect in the bodyof an animal. In formulating some pharmaceutical compositions describedherein, the compound is preferably soluble or dispersible in thenon-polymeric composition to form a homogeneous mixture, and uponimplantation, becomes incorporated into the implant matrix. As the solidmatrix degrades over time, the compound is capable of being releasedfrom the matrix into the adjacent tissue fluid, and to the pertinentbody tissue or organ, either adjacent to or distant from the implantsite, preferably at a controlled rate. The release of the compound fromthe matrix may be varied by the solubility of the compound in an aqueousmedium, the distribution of the compound within the matrix, the size,shape, porosity, and solubility and biodegradability of the solidmatrix. See e.g., U.S. Pat. No. 5,888,533. The amounts andconcentrations of ingredients in the composition administered to thepatient will generally be effective to accomplish the task intended.

In other aspects, the compounds of the present invention may beadministered by bioactive agent delivery systems containingmicroparticles suspended in a polymer matrix. The microparticles may bemicrocapsules, microspheres or nanospheres currently known in the art.The microparticles should be capable of being entrained intact within apolymer that is or becomes a gel once inside a biological environment.The microparticles can be biodegradable or non-biodegradable. Manymicroencapsulation techniques used to incorporate a bioactive agent intoa microparticle carrier are taught in the art. See e.g., U.S. Pat. Nos.4,652,441; 5,100,669; 4,438,253; and 5,665,428.

A preferred polymeric matrix will be biodegradable and exhibit watersolubility at low temperature and will undergo reversible thermalgelation at physiological mammalian body temperatures. The polymericmatrix is capable of releasing the substance entrained within its matrixover time and in a controlled manner. The polymers are graduallydegraded by enzymatic or non-enzymatic hydrolysis in aqueous orphysiological environments. See e.g., U.S. Pat. No. 6,287,588.

Methods of Preparation

Methods of preparing various pharmaceutical compositions with a certainamount of active ingredients are known, or will be apparent in light ofthis disclosure, to those skilled in the art. Methods of preparing saidpharmaceutical compositions can incorporate other suitablepharmaceutical excipients and their formulations as described inREMINGTON'S PHARMACEUTICAL SCIENCES, Martin, E. W., ed., Mack PublishingCompany, 19th ed. (1995).

Pharmaceutical preparations of the present invention are manufactured ina manner that is known, including conventional mixing, dissolving, orlyophilizing processes. Thus, liquid pharmaceutical preparations can beobtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary.

One of ordinary skill in the art will appreciate that a method ofadministering pharmaceutically effective amounts of the compositions ofthe invention to a patient in need thereof, can be determinedempirically, or by standards currently recognized in the medical arts.The agents can be administered to a patient as pharmaceuticalcompositions in combination with one or more pharmaceutically acceptableexcipients. It will be understood that, when administered to a humanpatient, the total daily usage of the agents of the compositions of thepresent invention will be decided within the scope of sound medicaljudgment by the attending physician. The specific therapeuticallyeffective dose level for any particular patient will depend upon avariety of factors: the type and degree of the cellular response to beachieved; activity of the specific agent or composition employed; thespecific agents or composition employed; the age, body weight, generalhealth, gender and diet of the patient; the time of administration,route of administration, and rate of excretion of the agent; theduration of the treatment; drugs used in combination or coincidentalwith the specific agent; and like factors well known in the medicalarts. It is well within the skill of the art to start doses of theagents at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosages until thedesired effect is achieved.

Dosaging can also be administered in a patient-specific manner toprovide a predetermined concentration of the agents in the blood, asdetermined by techniques accepted and routine in the art.

V. DOSAGE DETERMINATIONS

In general, the compounds disclosed herein may be used alone or inconcert with other therapeutic agents at appropriate dosages defined byroutine testing in order to obtain optimal efficacy while minimizing anypotential toxicity. The dosage regimen utilizing a compound of thepresent invention may be selected in accordance with a variety offactors including type, species, age, weight, sex, medical condition ofthe patient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound employed. A physician or veterinarian of ordinaryskill can readily determine and prescribe the effective amount of thedrug required to prevent, counter, or arrest the progress of thecondition.

Optimal precision in achieving concentrations of drug within the rangethat yields maximum efficacy with minimal toxicity may require a regimenbased on the kinetics of the compound's availability to one or moretarget sites. Distribution, equilibrium, and elimination of a drug maybe considered when determining the optimal concentration for a treatmentregimen. The dosages of a compound disclosed herein may be adjusted whencombined to achieve desired effects. On the other hand, dosages of thesevarious therapeutic agents may be independently optimized and combinedto achieve a synergistic result wherein the pathology is reduced morethan it would be if either agent were used alone.

In particular, toxicity and therapeutic efficacy of a compound disclosedherein may be determined by standard pharmaceutical procedures in cellcultures or experimental animals, e.g., for determining the LD₅₀ (thedose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index and it maybe expressed as the ratio LD₅₀/ED₅₀. Compounds exhibiting largetherapeutic indices are preferred except when cytotoxicity of thecompound is the activity or therapeutic outcome that is desired.Although compounds that exhibit toxic side effects may be used, adelivery system can target such compounds to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects. Generally, the compounds of the present inventionmay be administered in a manner that maximizes efficacy and minimizestoxicity.

Data obtained from cell culture assays and animal studies may be used informulating a range of dosages for use in humans. The dosages of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compound used in the methods of thepresent invention, the therapeutically effective dose may be estimatedinitially from cell culture assays. A dose may be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (the concentration of the test compound that achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation may be used to accurately determine useful doses in humans.Levels in plasma may be measured, by high performance liquidchromatography.

Moreover, the dosage administration of the pharmaceutical compositionsof the present invention may be optimized using apharmacokinetic/pharmacodynamic modeling system. One or more dosageregimens may be chosen and a pharmacokinetic/pharmacodynamic model maybe used to determine the pharmacokinetic/pharmacodynamic profile of oneor more dosage regimens. Next, one of the dosage regimens foradministration may be selected which achieves the desiredpharmacokinetic/pharmacodynamic response based on the particularpharmacokinetic/pharmacodynamic profile. See U.S. Pat. No. 6,747,002,which is entirely expressly incorporated herein by reference.

Methods are known in the art for determining effective doses fortherapeutic and prophylactic purposes for the disclosed pharmaceuticalcompositions or the disclosed drug combinations, whether or notformulated in the same composition. For therapeutic purposes, the term“jointly effective amount,” as used herein, means that amount of eachactive compound or pharmaceutical agent, alone or in combination, thatelicits the biological or medicinal response in a tissue system, animalor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician, which includes alleviation of the symptoms ofthe disease or disorder being treated. For prophylactic purposes (i.e.,inhibiting the onset or progression of a disorder), the term “jointlyeffective amount” refers to that amount of each active compound orpharmaceutical agent, alone or in combination, that inhibits in asubject the onset or progression of a disorder as being sought by aresearcher, veterinarian, medical doctor or other clinician. Thus, thepresent invention further provides combinations of two or moretherapeutic agents wherein, (a) each therapeutic agent is administeredin an independently therapeutically or prophylactically effectiveamount; (b) at least one therapeutic agent in the combination isadministered in an amount that is sub-therapeutic or subprophylactic ifadministered alone, but is therapeutic or prophylactic when administeredin combination with the second or additional therapeutic agentsaccording to the invention; or (c) both therapeutic agents areadministered in an amount that is subtherapeutic or sub-prophylactic ifadministered alone, but are therapeutic or prophylactic whenadministered together. Combinations of three or more therapeutic agentsare analogously possible. Methods of combination therapy includeco-administration of a single formulation containing all active agents;essentially contemporaneous administration of more than one formulation;and administration of two or more active agents separately formulated.

More specifically, the pharmaceutical compositions may be administeredin a single daily dose, or the total daily dosage may be administered individed doses of two, three, or four times daily. Doses may beadministered for one week, one month, or over the course of severalmonths, 3, 6, 9 or 12 months, or intervals known in the art anddetermined to be clinically relevant. Doses may be continued throughoutthe life of the patient, or discontinues when clinical judgmentwarrants. The daily dosage of the compositions may be varied over a widerange from about 0.0001 to about 1,000 mg per patient, per day. Therange may more particularly be from about 0.001 mg/kg to 10 mg/kg ofbody weight per day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20mg per day for adults (at about 60 kg). Additionally, the dosages may beabout 0.5-10 mg/kg per day, about 1.0-5.0 mg/kg per day, 5.0-10 mg/kgper day, or equivalent doses as determine by a practitioner, to achievea serum concentration that is clinically relevant.

In the case of injections, it is usually convenient to give by anintravenous route in an amount of about 0.01-30 mg, about 0.1-20 mg orabout 0.1-10 mg per day to adults (at about 60 kg). Intravenous dosesmay include a bolus or a slow dosing. In the case of other animals, thedose calculated for 60 kg may be administered as well.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of a compound of the presentinvention 0.0001 to about 1,000 mg per patient, per day. The range maymore particularly be from about 0.001 mg/kg to 10 mg/kg of body weightper day, about 0.1-100 mg, about 1.0-50 mg or about 1.0-20 mg per dayfor adults (at about 60 kg). Additionally, the dosages may be about0.5-10 mg/kg per day, about 1.0-5.0 mg/kg per day, 5.0-10 mg/kg per day,or equivalent doses as determine by a practitioner, to achieve a serumconcentration that is clinically relevant.

Specifically, the pharmaceutical compositions of the present inventionmay be administered at least once a week over the course of severalweeks. In one aspect, the pharmaceutical compositions are administeredat least once a week over several weeks to several months. In anotheraspect, the pharmaceutical compositions are administered once a weekover four to eight weeks. In yet another aspect, the pharmaceuticalcompositions are administered once a week over four weeks.

VI. METHODS OF USE OF THE COMPOUNDS OF THE INVENTION

In another aspect, the present invention is further directed to methodsthat have utility in the treatment of any diseases associated withneuron loss. More specifically, the present invention further providesmethods for stimulating neurogenesis and/or inhibiting neuronaldegeneration in a mammal. In a specific aspect, the method may compriseadministering to a mammal a composition comprising a compound describedherein. The composition comprising a compound described herein may beadministered in an amount effective to stimulate neurogenesis and/orinhibit neuronal degeneration in the mammal.

In a further aspect, a method for treating a mammal afflicted with aneurodegenerative disease or condition may comprise administering aneffective amount of a composition comprising a compound described hereinto the mammal. In other aspects, the neurodegenerative disease orcondition may be selected from the group consisting of ischemic stroke,traumatic brain injury, acute disseminated encephalomyelitis,amyotrophic lateral sclerosis (ALS), retinitis pigmentosa, mildcognitive impairment, Alzheimer's disease, Pick's disease, seniledementia, progressive supranuclear palsy, subcortical dementias, Wilsondisease, multiple infarct disease, arteriosclerotic dementia, AIDSassociated dementia, cerebellar degeneration, spinocerebellardegeneration syndromes, Friedreichs ataxia, ataxia telangiectasia,epilepsy-related brain damage, spinal cord injury, restless legssyndrome, Huntington's disease, Parkinson's disease, striatonigraldegeneration, cerebral vasculitis, mitochondrial encephalomyopathies,neuronal ceroid lipofuscinosis, spinal muscular atrophies, lysosomalstorage disorders with central nervous system involvement,leukodystrophies, urea cycle defect disorders, hepatic encephalopathies,renal encephalopathies, metabolic encephalopathies, porphyria, bacterialmeningitis, viral meningitis, meningoencephalitis, prion diseases,poisonings with neurotoxic compounds, Guillain Barre syndrome, chronicinflammatory neuropathies, polymyositis, dermatomyositis andradiation-induced brain damage. Included in the aspect isneurodegeneration including peripheral neuropathy due to therapeuticadministration of cranial irradiation or chemotherapeutic agents.

In another aspect, a method for treating a mammal afflicted with aneuropsychiatric disease or condition may comprise administering aneffective amount of a composition comprising a compound described hereinto the mammal. In other aspects, the neuropsychiatric disease orcondition may be selected from the group consisting of anxietydisorders, childhood disorders, eating disorders, mood disorders,cognitive disorders, personality disorders, psychotic disorders, andsubstance-related disorders.

More specifically, the types of psychiatricdiseases/disorders/conditions that may be treated using the compounds ofthe present invention include anxiety disorders including, but notlimited to, acute stress disorder, panic disorder, agoraphobia, socialphobia, obsessive-compulsive disorder, posttraumatic stress disorder,and generalized anxiety disorder; childhood disorders including, but notlimited to, attention-deficit hyperactivity disorder, asperger'sdisorder, autistic disorder, conduct disorder, oppositional defiantdisorder, separation anxiety disorder, and tourette's disorder; eatingdisorders including, but not limited to, anorexia nervosa, and bulimianervosa; mood disorders including, but not limited to, major depressivedisorder, bipolar disorder (manic depression), cyclothymic disorder, anddysthymic disorder; cognitive disorders including, but not limited to,delirium, multi-infarct dementia, dementia associated with alcoholism,dementia of the alzheimer type, and dementia; personality disordersincluding, but not limited to, paranoid personality disorder, schizoidpersonality disorder, schizotypal personality disorder, antisocialpersonality disorder, borderline personality disorder, histrionicpersonality disorder, narcissistic personality disorder, avoidantpersonality disorder, dependent personality disorder, andobsessive-compulsive personality disorder; psychotic disordersincluding, but not limited to, schizophrenia, delusional disorder, briefpsychotic disorder, schizophreniform disorder, schizoaffective disorder,and shared psychotic disorder; substance-related disorders including,but not limited to, alcohol dependence, amphetamine dependence, cannabisdependence, cocaine dependence, hallucinogen dependence, inhalantdependence, nicotine dependence, opioid dependence, phencyclidinedependence, and sedative dependence.

In another aspect, a method for treating a mammal afflicted withtraumatic brain injury may comprise administering an effective amount ofa composition comprising a compound described herein to the mammal.

In another aspect, a method for treating a mammal afflicted withtraumatic nerve injury may comprise administering an effective amount ofa composition comprising a compound described herein to the mammal.

In another aspect, the invention includes a compound having a structureaccording to Formula I:

wherein R₁ is hydrogen, alkyl, branched alkyl, aryl, aralalkyl, benzyl,napthyl, cycloalkyl of 1 to 12 carbon atoms, or a heterocycle, when R₁is not hydrogen it can be optionally substituted with H, OH, alkyl,alkoxy, halogen, —CF₃, —R₂, —OR₂, —SR₂, —N(R₂)₂, —CN, —NO₂, —NC(O) R₂,—C(O)R₂, —C(O)N(R₂)₂, —S(O)₂R₂, —S(O)₂NR₂, —S(O)R₂, —C(O)R₂, —C(O)OR₂,or —C(O)N(R₂)₂ wherein each R₂ is independently H, alkyl, alkenyl,alkynyl, aryl, heterocycle, protecting group or prodrug moiety;

-   L is [CH₂]₂₋₆, —CH₂—(C═O)—CH₂—,

-   X is S, SO₂, O, or NH;-   Y is

-   Z is

each optionally substituted with H, OH, alkyl, alkoxy, halogen, or —CF₃;

-   T is O, S, or NR₁;-   n is 2 or 3;-   wherein each R₁ is independent and may be the same as each other or    preferably different.

The invention also includes a method for stimulating neurogenesis and/orinhibiting neuronal degeneration in a mammal, comprising administering apharmaceutical composition in an amount effective to stimulateneurogenesis and/or inhibit neuronal degeneration in the mammal, saidpharmaceutical composition comprising:

wherein R₁ is hydrogen, alkyl, branched alkyl, aryl, aralalkyl, benzyl,napthyl, cycloalkyl of 1 to 12 carbon atoms, or a heterocycle, when R₁is not hydrogen it can be optionally substituted with H, OH, alkyl,alkoxy, halogen, —CF₃, —R₂, —OR₂, —SR₂, —N(R₂)₂, —CN, —NO₂, —NC(O)R₂,—C(O)R₂, —C(O)N(R₂)₂, —S(O)₂R₂, —S(O)₂NR₂, —S(O)R₂, —C(O)R₂, —C(O)OR₂,or —C(O)N(R₂)₂ wherein each R₂ is independently H, alkyl, alkenylalkynyl, aryl, heterocycle, protecting group or prodrug moiety;

-   L is [CH₂]₂₋₆, —CH₂—(C═O)—CH₂—,

-   X is S, SO₂, O, or NH;-   Y is

-   Z is

each optionally substituted with H, OH, alkyl, alkoxy, halogen, or —CF₃;

-   T is O, S, or NR₁;-   n is 2 or 3;-   wherein each R₁ is independent and may be the same as each other or    preferably different a pharmaceutically acceptable carrier; and-   optionally wherein the pharmaceutical composition is administered to    a patient in need thereof, to treat a condition selected from the    group consisting of neurodegenerative disease, psychiatric disorders    and aging.

Also included is a method for stimulating neurogenesis and/or inhibitingneuronal degeneration in a mammal, comprising administering apharmaceutical composition in an amount effective to stimulateneurogenesis and/or inhibit neuronal degeneration in the mammal,additionally including wherein the pharmaceutical composition isadministered to a patient in need thereof, to treat a condition selectedfrom the group consisting of neurodegenerative disease, psychiatricdisorders and aging, the pharmaceutical compositions comprising acompound selected from the group consisting of:

and pharmaceutically acceptable salts, hydrates, or solvates thereof,and a pharmaceutically carrier.

The invention also includes a method wherein the pharmaceuticalcomprising any one or a combination thereof, of the compositionsdescribed herein is administered to a patient in need thereof, to treata condition including neurodegnerative disease, psychiatric disorders,brain injury, nerve injury and/or aging.

The invention is also directed to methods of stimulating neurogenesisand/or inhibiting neurodegeneration in vitro and ex vivo. Examples of invitro uses include, but are not limited to, stimulating growth ofneurons in cultured cells and tissue, for example, muscle, skin, bone,cartilage, ligament, tendon, tooth, eye, brain, spinal cord, heart,blood vessel, lymph node, ovary, oviduct, uterus, vagina, mammary gland,testicular, seminal vesicle, penis, hypothalamus, pituitary, thyroid,pancreas, adrenal gland, kidney, ureter, bladder, urethra, mouth,esophagus, stomach, small intestine, large intestine, salivary gland,taste bud, nasal, trachea, and lung tissue. Non-limiting examples of exvivo uses include inhibiting neurodegeneration or stimulatingneurogenesis in organ tissues, including but not limited to intactorgans and organ systems such as muscle, skin, bone, cartilage,ligament, tendon, tooth, eye, brain, spinal cord, heart, blood vessel,lymph node, ovary, oviduct, uterus, vagina, mammary gland, testicular,seminal vesicle, penis, hypothalamus, pituitary, thyroid, pancreas,adrenal gland, kidney, ureter, bladder, urethra, mouth, esophagus,stomach, small intestine, large intestine, salivary gland, taste bud,nasal, trachea, and lung tissue. In addition, the compositions andmethods of the invention are useful in conjunction with pluripotent andmultipotent stem cells, including but not limited to adult neural stemcells, which retain the capacity to differentiate into a wide range ofneurons and glia. Neurons derived from such neural stem cells arecapable of migrating to various regions of the CNS, receiving afferentinnervation, forming axonal projections, and expressingneurotransmitters. The compositions of the invention may be used aloneto promote neurogenesis of neural stem cells in vitro, in vivo and exvivo, and may also be advantageously used in combination with knowngrowth factors including, but not limited to fibroblast growth factors(FGFs), epidermal growth factors (EGFs), transforming growth factors(TGFs) and/or neurotrophic factors, non-limited examples of whichinclude brain-derived neurotrophic growth factor (BDNF) and ciliaryneurotrophic factor (CNTF). Accordingly, the compositions and methods ofthe invention are useful in regenerative medicine, in which stem cellsare coaxed into forming cells of any lineage (including neural cells)and then delivered to regions of injury or degeneration to treatdisease. Use of the compositions and methods of the invention isadvantageous in conjunction with growth factors to direct endogenousneural stem cells to differentiate into neurons or glia, as well as incell-replacement therapies based on delivery of ex vivo-derived neuralcells to areas of injury or degeneration.

The invention also includes compounds and compositions wherein thecompounds of the invention are present in a salt form. Examples of saltsinclude basic nitrogen-containing bisphosphonic acid salts, ammoniumsalts, alkali metal salts such as potassium and sodium (including butnot limited to mono-, di- and tri-sodium) salts, alkaline earth metalsalts such as calcium, magnesium and manganese, salts with organic basessuch as dicyclohexylamine salts, N-methyl-D-glucamine, and salts withorganic amino acids such as arginine, lysine or histadine. Non-toxic,physiologically acceptable salts are preferred.

The invention also includes a kit comprising two or more containers,having a first container containing a therapeutically effective amountof the pharmaceutical composition comprising any one of Formulas I toXII, and a second container comprising a carrier, excipient or diluent,and/or wherein a third container comprises a therapeutically-acceptableamount of an additional therapeutically active agent. The kit alsocomprises the composition comprising any one of Formulas I to XII,standardized research grade reagents and control standards and also cancomprise two or more compositions comprising a compound of any ofFormulas I to XII.

The invention includes a method to promote neurogenesis in a mammaliancell including neural stem cells, embryonic stem cells and progenitorcells, said method comprising:

-   culturing the cells in a cell medium, in the presence of the    composition comprising any of Formulas I to XII, and observing said    cells for expression of neurogenesis, including greater cell    numbers, quality of the cells, differentiation of cells, or a    combination thereof.

Other objectives, features and advantages of the present invention willbecome apparent from the following specific examples. The specificexamples, while indicating specific aspects of the invention, areprovided by way of illustration only. Accordingly, the present inventionalso includes those various changes and modifications within the spiritand scope of the invention that may become apparent to those skilled inthe art from this detailed description. The invention will be furtherillustrated by the following non-limiting examples.

EXAMPLES Example 1 Identification of Compounds

The in vitro neurogenesis test was performed using human neuronalprogenitors cells. By way of background, the human neuronal progenitorcells can be cultured in media and have the potential to produce maturefunctional neurons. The neurogenesis factor in the culture media canpromote the number of the progenitor cells which differentiate intoneurons. Indeed, it is a widely accepted in vitro model to test achemical's neurogenetic property.

Cells were obtained as human neuronal progenitors from a commercialsource and were grown for up to three passages or at any timedifferentiated to potentially produce mature functional neurons inculture. Following two passages to expand the cells, progenitors wereseeded into multi-well microplates and the media were changed todifferentiation media (minus serum and mitogen). Within 2 hrs of thismedia change, a vehicle, positive control or test compound were added toeach well. Also, they were added with every further media change (50%volume change every other day). Control wells contained cells andvehicle (0.2% DMSO in DMEM/F12). Other wells contained positive controlsfor neuronal progenitor growth, leukemia inhibitory factor (LIF, 10ng/ml).

The cells were stained using MAP-2, a neuronal marker, on day 11. Then,each test compound was assessed for the ability to promote an increasein neuron number and compared with the positive control, leukemiainhibitory factor (LIF).

The data in Table 2 demonstrates that examples of the compounds of thepresent invention showed higher neurogenesis activity than the positivecontrol in the in vitro neurogenesis test. That is, the cells given thesubject compound showed a further increased number of neurons ascompared to the cells given the positive control.

TABLE 2 Effects of compounds on the increase in the neuron numberscompared with the vehicle control Compound % of Control3-(4-Methoxyphenyl)-6[(1-ethylpyrrolidin-2-yl)methyl]- 107isothiazolo[4,5-d]pyrimidin-7(6H)-one3-(4-Methoxyphenyl)-6-[3-(4-morpholinyl)propyl]- 115isothiazolo[4,5-d]pyrimidin-7(6H)-one

Example 2 Examination of Compounds Effectiveness to Inhibit NeuronalDegeneration

The effective compounds were then subjected to the in vitro neurondegeneration test. This test as applied for neuroprotective discoveryinvolved the ability of the drugs to inhibit apoptosis and necrosis.

In measuring the ability of inhibiting apoptosis, mature neurons (3-4weeks following initiation of differentiation) were treated withstaurosporine to induce apoptosis. A low concentration of staurosporine(10-100 nM) or beta amyloid 1-42 peptide at 1-10 μM or peptide betaamyloid 25-35 at 10-75 μM concentration was used to stimulate apoptosis.At the same time as treating with staurosporine, the neurons weretreated with vehicle or one of the test agents. Because there was strongdata demonstrating that staurosporine activated caspase-3 to initiatethe apoptotic pathway, the compounds of the invention abilities ofinhibiting staurosporine-induced apoptosis were quantified by the amountof activated caspase-3. Each compound's inhibitory ability was comparedwith vehicle and staurosporine.

Necrosis of the mature neurons was initiated using beta amyloid 1-42peptide at 1-10 μM or peptide beta amyloid 25-35 at 10-75 μMconcentration. This synthetic peptide was of the same length naturallyfound in AD brain. Because lactate dehydrogenase (LDH) is released fromcells when the plasma membrane is impaired, the cell loss was quantifiedby the amount of LDH released into the media following a 24-48 hrtreatment. The ability of neurogenic agents to reduce the LDH releaseinduced by beta amyloid, versus vehicle control was used as themeasurement of inhibition of neuron degeneration.

Dysfunctional neurons were initiated using any of the above agents orusing hydrogen peroxide at 1-100 μM concentration. Using a dye thatmeasures metabolic activity of the cells, such as MTT or ALAMARBLUE®, wedetermined the reduction of respiratory capacity of the cells, whichindicated neuron dysfunction. The ability of neurogenic agents toinhibit hydrogen peroxide-induced reduction in cellular respiration wasused as a measure of inhibition of neuronal dysfunction, a potentialstep that lead to degeneration.

Then the compounds were subjected to a series of tests to determine theunderlining mechanism of their neurogenesis and neuroprotectiveeffectiveness as well as their toxicities. Examples of receptors thatwere examined in this screen were: adenosine, adrenergic, cannabinoid,dopamine, GABA, glutamate, histamine, muscarinic, opiate receptors aswell as calcium, potassium and sodium channels. Then, the compounds weretested in a HERG cellular test to identify their potential cardiactoxicity, an Ames test to identify their mutagenicity, and an Irwinscreen to identify their central nervous system toxicity.

The compounds' efficacies were evaluated in the in vivo Behavior AgingModel test and a transgenic models of Alzheimer's test. In the BehaviorAging Model, the compounds were assessed by measuring the ability ofproducing improvement in cognitive function of motor skills of21-month-old C57BL/6 mice. The ability of C57BL/6 mice to learn thewater maze task has been previously shown to decline with age (e.g.,Fordyce and Wehner, 1993; Frick et al., 2000; Forster et al., 1996;Sumien et al., 2004; McDonald & Forster 2005, in press), and isassociated with the degree of hippocampal neurogenesis (Kempermann etal., 2002) and with functional indicators of hippocampal synapticplasticity (van Praag et al., 1999). A series of behavior tests thatrelate to the learning of an active avoidance response, the measurementof reaction time, the memory for habituation, the age-sensitive measuresof motor skills, spontaneous ambulation, and sensory function werecarried out to assess the scope and specificity of behavior improvement.

Certain compounds were further analyzed to determine their ability toinhibit neurodegeneration by hydrogen peroxide. Human neurons aretreated with hydrogen peroxide (50 uM) for up to 6 hrs and then neuronalloss is measured using Alamar Blue respiration. If either NNI-A (FormulaX) or NNI-B (Formula XI) is added along with hydrogen peroxide less lossof respiration is observed. These results are demonstrated in FIG. 1.

Compounds for testing were chosen to have fair blood brain barrierpenetration (Log_(BB)>−0.3) and also have drug-like properties, i.e.,they follow “Lipinki's Rule of Five” (See, C A Lipinski, Adv. Drug Del.Rev. 1997, 23, 3). The “Rule of 5” states that: poor absorption orpermeation is more likely when:

1. There are more than 5 H-bond donors (expressed as the sum of OHs andNHs).

2. The Molecular Weight is over 500.

3. The Log_(P) is over 5 (or MLogP is over 4.15).

4. There are more than 10 H-bond acceptors (expressed as the sum of Nsand Os).

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any aspectthereof. All patents and publications cited herein are fullyincorporated by reference in their entirety.

What is claimed is:
 1. A compound selected from the group consisting of:

and pharmaceutically acceptable salts, hydrates, or solvates thereof. 2.A pharmaceutical composition comprising a compound selected from thegroup consisting of:

and pharmaceutically acceptable salts, hydrates, or solvates thereof.